The vertebrate immune system established different ways to detect invading pathogens based on certain characteristics of their microbial nucleic acids. Detection of microbial nucleic acids alerts the immune system to mount the appropriate type of immune response that is required for the defense against the respective type of pathogen detected. Detection of viral nucleic acids leads to the production of type I interferon (IFN), the key cytokine for anti-viral defense. An understanding of how nucleic acids interact with the vertebrate immune system is important for developing different nucleic acid-based therapeutic strategies for the immunotherapy of diseases (Rothenfusser S et al. 2003, Curr Opin Mol Ther 5:98-106) and for developing gene-specific therapeutic agents (Tuschl T et al. 2002, Mol Interv 2: 158-167).
For the recognition of long dsRNA, two detection modes are known, the serine threonine kinase PKR (Williams B R, 2001, Sci Signal Transduction Knowledge Environment 89: RE2; Meurs E F et al. 1992, J Virol 66: 5805-5814; Katze M G et al. 1991, Mol Cell Biol 11: 5497-5505) and Toll-like receptor (TLR) 3 (Alexopoulou L et al. 2001, Nature 413: 732-738). Whereas PKR is located in the cytosol, TLR3 is present in the endosomal compartment (Matsumoto M et al. 2003, J Immunol 171: 3154-3162). TLR3 is a member of the Toll-like receptor family that has evolved to detect pathogen-specific molecules (Takeda K et al. 2003, Annu Rev Immunol 21: 335-376).
A second characteristic feature of viral nucleic acids used by the immune system to recognize viral infection are CpG motifs found in viral DNA, which are detected via TLR9 (Lund J et al. 2003, J Exp Med 198: 513-520; Krug A et al. 2004, Blood 103: 1433-1437). CpG motifs are unmethylated CG dinucleotides with certain flanking bases. The frequency of CpG motifs is suppressed in vertebrates, allowing the vertebrate immune system to detect microbial DNA based on such CpG motifs (Krieg A M et al. 1995, Nature 374: 546-549; Bauer S et al. 2001, Proc Natl Acad Sci USA 98: 9237-9242; Wagner H et al. 2002, Curr Opin Microbiol 5: 62-69). Like TLR3, TLR9 is located in the endosomal compartment where it directly binds to CpG motifs (Latz E et al. 2004, Nat Immunol 5: 190-198).
In addition to long dsRNA and CpG DNA, two recent publications suggest a third mechanism by which viral nucleic acids are recognized. These studies demonstrate that single-stranded RNA (ssRNA) of ssRNA viruses is detected via TLR7 (mouse and human) and TLR8 (only human) (Diebold S S et al. 2004, Science 303: 1529-1531; Heil F et al. 2004, Science 303: 1526-1529). Guanine analogues have been identified earlier as specific ligands for TLR7 and TLR8 (Lee J et al. 2003, Proc Natl Acad Sci USA 100: 6646-6651; Heil F et al. 2003, Eur J Immunol 33: 2987-2997). Like TLR9 (receptor for CpG DNA) (Latz E et al. 2004, Nat Immunol 5: 190-198), TLR7 and TLR8 are located in the endosomal membrane (Heil F et al. 2003, Eur J Immunol 33: 2987-2997).
Detection of viral nucleic acids leads to the production of type I IFN (IFN-α and IFN-β). The major producer of type I IFN in humans is the plasmacytoid dendritic cell (PDC, also called interferon producing cell, IPC). The plasmacytoid dendritic cell (PDC) is a highly specialized subset of dendritic cells that is thought to function as a sentinel for viral infection and is responsible for the vast amount of type I IFN during viral infection (Asselin-Paturel C et al. 2001, Nat Immunol 2: 1144-1150). There is increasing evidence that PDC preferentially use nucleic acid-based molecular patterns to detect viral infection. TLR expression of human and mouse PDC is limited to TLR7 and TLR9 (Krug A et al. 2001, Eur J Immunol 31: 3026-3037; Hornung V et al. 2002, J Immunol 168: 4531-4537; Edwards A D et al. 2003, Eur J Immunol 33: 827-833).
IFN-α was the first type of interferon to be identified and commercialized; it is widely used clinically in the treatment of a variety of tumors (e.g., hairy cell leukemia, cutaneous T cell leukemia, chronic myeloid leukemia, non-Hodgkin's lymphoma, AIDS-related Kaposi's sarcoma, malignant melanoma, multiple myeloma, renal cell carcinoma, bladder cell carcinoma, colon carcinoma, cervical dysplasia) and viral diseases (e.g., chronic hepatitis B, chronic hepatitis C). IFN-α products that are currently in clinical use include the recombinant protein and the highly purified natural protein, both of which have high production costs. Therefore, there is a need for more economical ways of providing IFN-α to patients in need. Furthermore, IFN-α is currently administrated systematically and causes a broad spectrum of side effects (e.g. fatigue, flu-like symptoms, diarrhea). Most alarmingly, IFN-α causes a decrease in bone marrow function which leads to increased susceptibility to life-threatening infections, anemia and bleeding problems. Therefore, there is a need for ways of providing IFN-α in a more localized (i.e., target-specific) matter to reduce the occurrence of side effects.
In addition to inducing an anti-viral interferon response, dsRNA also induces post-transcription gene silencing, a highly conserved anti-viral mechanism known as RNA interference (RNAi). Briefly, the RNA III Dicer enzyme processes dsRNA into short interfering RNA (siRNA) of approximately 22 nucleotides. The antisense strand of the siRNA binds a target mRNA via base pairing and serves as a guide sequence to induce cleavage of the target mRNA by an RNA-induced silencing complex RISC. dsRNA has been an extremely powerful tool in studying gene functions in C. elegence and Drosophila via gene silencing. However, its use in mammalian cells has been limited because the interferon response it elicits is detrimental to most mammalian cells.
Subsequently, it was found that siRNA was also capable of inducing RNAi, causing degradation of the target mRNA in a sequence-specific manner and it was thought to be short enough to bypass dsRNA-induced nonspecific effects in mammalian cells (Elbashri S M et al. 2001, Nature 411:494-498). Since then, siRNA has been widely used as a gene silencing tool in deciphering mammalian gene functions in research and drug discovery, and there has been great interest in its potential in therapeutic applications.
siRNA can be used to reduce or even abolish the expression of disease/disorder-related genes for preventing or treating diseases caused by the expression or overexpression of the disease-related genes. Such diseases include, but are not limited to, infections, metabolic diseases, autoimmune diseases and cancer. However, concern has been raised recently about the potential for siRNA to activate immune responses which may be undesirable for certain indications and thus limit the use of siRNA as a gene silencing agent for therapeutic purposes (Sioud M et al. 2003, Biochem. Biophys. Res. Commun. 312:1220-1225). Therefore, there is a need for methods for predicting the potential of a given siRNA to induce an interferon response and for methods for designing and preparing siRNAs for gene silencing which are devoid of unwanted immunostimulatory activities.
On the other hand, for certain therapeutic applications, for example, the prevention or treatment of cancer and viral infections, immunostimulatory activity may be desirable as an additional functional activity of the siRNA.
In an effort to apply siRNA for the specific downregulation of TLR9 in PDC in our previous publication (Hornung V et al. 2005, Nat Med 11: 263-270), we made the surprising observation that, despite the inability of PDC to detect long dsRNA, certain siRNA sequences were potent in vitro inducers of IFN-α in PDC. We found that i) short interfering RNA (siRNA) induces IFN-α in human plasmacytoid dendritic cells when transfected with cationic lipids, ii) this activity of siRNA is sequence-dependent but independent of the G or U content of the siRNA, iii) the immunostimulatory activity of siRNA and the antisense activity are two independent functional activities of siRNA, iv) the immune recognition of siRNA occurs on the single strand level, v) siRNAs containing the 9mer sequence motif 5′-GUCCUUCAA-3′ show potent immunostimulatory activity, and vi) such siRNAs induce systemic immune responses in mice, and vii) the induction of immune responses by siRNA requires the presence of TLR7 in mice. Our findings suggest that the 9mer sequence motif 5′-GUCCUUCAA-3′ may be a ligand for TLR7.
The natural ligand for TLR7 has not been well defined to date. Guanine analogues have been identified earlier as specific ligands for TLR7 and TLR8 (Lee J et al. 2003, Proc Natl Acad Sci USA 100: 6646-6651; Heil F et al. 2003, Eur J Immunol 33: 2987-2997), whereas guanosine ribonucleoside or a derivative thereof has been identified as TLR7 ligand in WO03086280.
It is an object of the present invention to identify RNA oligonucleotide motifs for stimulating an immune response, in particular, IFN-α induction. It is also an object of the present invention to identify ligands for activating TLR7 and TLR8. It is another object of the present invention to develop a method for determining the immunostimulatory activity, in particular, the IFN-α-inducing activity, of a RNA oligonucleotide. It is yet another object of the present invention to develop a method for predicting the immunostimulatory activity, in particular, IFN-α-inducing activity, of a RNA oligonucleotide. It is a further object of the invention to develop a method for designing and preparing RNA oligonucleotide having or lacking immunostimulatory activity, in particular, IFN-α-inducing activity. It is also an object of the invention to provide RNA oligonucleotides having high immunostimulatory activity which can be used to induce an immune response, in particular, IFN-α production, in patients in need thereof. It is yet another object of the present invention to provide siRNA molecules that either have or lack immunostimulatory activity which can be used to treat disorders caused by the expression or overexpression of disorder-related genes.